p53 is a tumor suppressor protein that regulates the expression of certain genes required for cell cycle arrest or apoptosis. The tumor suppressor gene encoding p53 is activated by DNA damage, cell stress, or the aberrant expression of certain oncogenes (Levine 1997). Once activated, wild type p53 (wt p53) serves to temporarily arrest the cell cycle, allowing time for DNA repair and preventing cells with damaged DNA from proliferating uncontrollably (Levine 1997). p53 is also involved in inducing apoptosis in cells with certain types of physiologic damage (Levine 1997).
Mutations in p53 that functionally inactivate its growth suppressing ability have been observed in 40-60% of all human cancers, and are associated with the malignant phenotype (Hainaut 2000). Mutations to p53 occur as early events in tumorigenesis (Millikan 1995; Querzoli 1998; Allred 1993), abrogating the ability of the protein to suppress cell division (Finlay 1989; Eliyahu 1989). The regulation of p53 expression in cells can occur at the level of p53 mRNA abundance or at the level of p53 protein abundance. Mutations of p53 are often associated with high nuclear and cytoplasmic concentrations of the p53 protein, due to the prolonged half-life of the mutated protein. Many tumors are characterized by highly elevated intracellular p53 levels compared to nonmalignant cells. Other tumors synthesize large amount of mutated p53, but contain low or below normal steady-state levels of intracellular p53, presumably as a result of accelerated intracellular degradation of the protein. Overexpression of p53 is an independent predictor of more aggressive cancers (Turner 2000; Elkhuizen 2000; Zellars 2000), lymph node metastases (Pratap 1998), failure to respond to standard therapies (Berns 1998; Berns 2000), and mortality (Sirvent 2001; Querzoli 2001).
Missense point mutations are the most frequent p53 mutations in cancer, leaving the majority of the p53 protein in its wild type form (wt p53). Although p53 mutations may represent true tumor specific antigens, most of these mutations occur at sites that do not correspond to immunologic epitopes recognized by T cells (Wiedenfeld 1994). Because of this, any widely applicable p53-directed immunotherapy must target wt p53. In experimental models, it has been possible to target p53 because the mutated molecule is associated with high nuclear and cytoplasmic concentrations of the p53 protein (Finlay 1988). p53 is an attractive target for adaptive immune response because the intracellular concentration of nonmutated p53 in healthy cells is very low (Zambetti 1993; Reich 1984). This means that healthy cells expressing non-mutant p53 will most likely escape an enhanced immune response to over-expressed mutant p53 (Offringa 2000).
p53, like most tumor associated antigens that are recognizable by the cellular arm of the immune system, is an autoantigen (Rosenberg 2001). The fact that p53 is an autoantigen widely expressed throughout development (Schmid 1991), coupled with the fact that the majority of mutated p53 being expressed in tumors has the same structure as the wild type protein, means that tumor-expressed p53 is likely to be tolerated as a self-protein by the immune system. This tolerance, which has been shown by functional and tetramer studies in mice to exist at the cytotoxic T lymphocyte level (CTL) (Theobald 1997; Erdile 2000), limits the effectiveness of p53-directed immunotherapies. To be successful, an effective immunotherapy must overcome this tolerance without also inducing autoimmunity against normal cells and tissues (Theobald 1997; Erdile 2000; Hernandez 2000). Small numbers of self-reacting T cells escape during the processes involved in the immune tolerance.
Tumors overexpressing p53 have been eliminated in murine models by the systemic administration of epitope specific CTL (Vierboom 2000a; Vierboom 2000b; Vierboom 1997; Hilburger 2001), epitope pulsed dendritic cells (DC) (Mayordomo 1996), or mutant p53 epitope with IL-12 (Noguchi 1995). Each of these strategies has considerable drawbacks with regards to clinical applicability. CTL infusion and infusion of epitope pulsed dendritic cells are time consuming and expensive, because the isolation, culturing, and reinfusion of cells must be performed individually for each patient. Conversely, in order to produce any effect, the cell-free vaccination strategies previously used required either intratumoral injections or vaccination prior to tumor challenge, neither of which represents a practical approach in the clinical setting. There is thus a need for simplified, efficient, and widely applicable immunotherapeutic strategies in the treatment of cancer.